Process for the detection of reverse transcriptase

Abstract

An ultrasensitive procedure for the detection of reverse transcriptase (RT) is proposed, which is divided into three steps:In the first step the sample is processed by placing a template-primer combination in a reaction mixture and using the RT activity present in the pretreated sample to provide a nucleic acid to be amplified in a given nucleic acid amplification procedure so as to ensure that this is only provided if there is RT activity in the sample.In the second step the nucleic acid to be amplified is amplified, the result being an amplification product.In the third step the amplification product is analyzed and identified.Compared to conventional RT assays a several million fold increase of sensitivity is achieved, thus the ultrasensitive detection of RT activity is made possible, that is the detection of all retroviruses as well as other retroelements containing or expressing active RT.The procedure is of importance for the diagnosis of infections and diseases caused by these in humans, animals and plants. Screening and typing kits are suggested for research and diagnostics.

Claims

1. A method for detection of reverse transcriptase (RT) in a sample comprising:

(a) pretreating the sample to make the total amount of reverse transcriptase contained therein available for a RT reaction; and eliminating from said sample factors which interfere with the RT reaction;

(b) incubating the pretreated sample with a template-primer combination in a reaction mixture, said template-primer combination comprising a template nucleic acid and at least a RT primer;

said RT primer being a nucleic acid which contains at least one functional sequence at least one of which is a hybridization sequence which extends to the 3'-end and by which the RT primer is hybridized to the template nucleic acid;

said template nucleic acid being a heteropolymeric nucleic acid which contains at least one segment consisting of a heteropolymeric RNA which is located upstream of a hybridization sequence to which said RT primer is hybridized and which is capable of functioning as a template in a RT reaction, said template nucleic acid further containing at least one additional functional sequence located upstream of said hybridization sequence for the RT primer;

(c) synthesizing a cDNA by reverse transcription catalyzed by said reverse transcriptase from the sample;

(d) amplifying by an in vitro nucleic acid amplification reaction at least part of the sequence of a cDNA produced in step (c) or of a nucleic acid which has been derived from said cDNA;

(e) analyzing and identifying the amplification product of step (d); and

(f) correlating the presence of amplification product to the presence of reverse transcriptase activity in the pretreated sample.

2. A method according to claim 1, wherein said template-primer combination incubated in step (b) contains all functional sequences which will be required to amplify at least part of the sequence of a cDNA synthesized in step (c); and in step (d) that part of the cDNA sequence which contains these functional sequences is amplified.

3. A method according to claim 2, wherein said template-primer combination of step (b) contains a pair of non-overlapping hybridization sequences which will be used in the amplification reaction of step (d), at least one of these being located upstream of that hybridization sequence of the template nucleic acid to which the RT primer is hybridized; a cDNA synthesized in step (c) contains said pair of hybridization sequences; and in step (d) part of the sequence of said cDNA is selectively amplified by a polymerase chain reaction using two amplification primers which anneal to said pair of hybridization sequences.

4. A method according to claim 3, wherein the template nucleic acid is the genomic RNA of the bacteriophage MS2, the RT primer is a synthetic deoxyoligonucleotide of the base sequence 5'-d(CATAGGTCAAACCTCCTAGGAATG)-3' (SEQ ID NO:1); and part of the MS2 cDNA synthesized in step (c) is selectively amplified in step (d) by polymerase chain reaction with synthetic deoxyoligonucleotide primers of the base sequence 5'-d(CATAGGTCAAACCTCCTAGGAATG)-3' (SEQ ID NO.: 1) and 5'-d(TCCTGCTCAACTTCCTGTCGAG)-3' (SEQ ID NO.: 2).

5. A method according to claim 2, wherein said template-primer combination of step (b) contains a pair of hybridization sequences immediately adjacent to each other which will be used in the amplification reaction of step (d), at least one of these being located entirely upstream of that hybridization sequence of the template nucleic acid to which the RT primer is hybridized; a cDNA synthesized in step (c) contains said pair of hybridization sequences; and in step (d) part of the sequence of said cDNA is selectively amplified by ligase chain reaction using said pair of hybridization sequences.

6. A method according to claim 2 wherein said template-primer combination of step (b) contains a pair of hybridization sequences which will be used in the amplification reaction of step (d), the hybridization sequences of said pair being separated from each other by a spacer sequence and at least one of them being located entirely upstream of that hybridization sequence of the template nucleic acid to which the RT primer is hybridized; a cDNA synthesized in step (c) contains said pair of hybridization sequences; and in step (d) part of the sequence of said cDNA is selectively amplified by a combination of a polymerase chain reaction and a ligase chain reaction using said pair of hybridization sequences.

7. A method according to claim 2, wherein said template-primer combination of step (b) contains a pair of non-overlapping hybridization sequences which will be used in the amplification reaction of step (d), at least one of these being located entirely upstream of that hybridization sequence of the template nucleic acid to which the RT primer is hybridized;

said cDNA synthesized in step (c) contains said pair of hybridization sequences;

in step (d) that part of said cDNA synthesized in step (c) which contains said pair of hybridization sequences is selectively amplified by

(i) employing a multienzymatic transcription-based procedure which uses said pair of hybridization sequences and reaction conditions that maintain the stability of the RNA; and

(ii) excluding the template RNA from amplification by prior treatment with at least one method selected from the group consisting of enzymatic degradation, chemical degradation, physical degradation, and dissociating the template RNA from a solid-phase-bound cDNA and washing it away.

8. A method according to claim 2, wherein said template-primer combination of step (b) contains a pair of non-overlapping hybridization sequences and a transcription promoter sequence which will be used in the amplification reaction of step (d), at least one hybridization sequence of said pair being located entirely upstream of that hybridization sequence of the template nucleic acid to which the RT primer is hybridized;

said cDNA synthesized in step (c) contains the P(-) sequence of said transcription promoter located upstream of said pair of hybridization sequences;

in step (d) that part of said cDNA synthesized in step (c) which contains said pair of hybridization sequences and said P(-) sequence is selectively amplified by

(i) employing a multienzymatic transcription-based procedure using said pair of hybridization sequences and said P(-) sequence under reaction conditions that maintain the stability of the RNA, and by

(ii) excluding the template RNA from amplification by prior treatment with at least one method selected from the group consisting of enzymatic degradation, chemical degradation, physical degradation, and dissociating the template RNA from a solid-phase-bound cDNA and washing it away.

9. A method according to claim 2, wherein the template-primer combination of step (b) contains all sequence elements of a replicative DNA; said template nucleic acid comprises, in 5' to 3' order, a DNA-ori(-) sequence which extends to the 5'-end and consists of a nucleic acid selected from the group comprising RNA and DNA, a spacer sequence, and a primer hybridization sequence which contains at least part of a DNA-ori(+) sequence extending to the 3'-end of said primer hybridization sequence;

said RT primer is a single-stranded DNA oligonucleotide which is in its entire length hybridized to at least the 3' terminal part of said DNA-ori(+) sequence of said template nucleic acid;

said cDNA synthesized in step (c) is a primary replicative DNA which contains a DNA-ori(-) sequence which extends to the 5'-end, a spacer sequence, and a DNA-ori(+) sequence which extends to the 3'-end, the hybridization of said 3' terminal DNA-ori(+) sequence to a 5' terminal DNA-ori(-) sequence constituting an active DNA-ori;

in step (d) said cDNA is amplified by

i) eliminating the template RNA at least partially by at least one method selected from the group comprising enzymatic degradation, chemical degradation, physical degradation, and dissociating the template RNA from a solid-phase-bound cDNA and washing it away, and

(ii) performing in a reaction mixture which contains dNTPs, a DNA replication procedure which involves binding of a protein primer to said active DNA-ori and replicating said replicative DNA by means of a DNA replicase that binds to said protein-primed replicative DNA.

10. A method according to claim 1, wherein said template nucleic acid of step (b) is RNA and contains at least one further hybridization sequence located upstream of that to which said RT primer is hybridized; said RT primer carries a functional group; a cDNA synthesized in step (c) contains said functional group and said at least one further hybridization sequence; a nucleic acid amplified in step (d) is derived from said cDNA by eliminating the template RNA at least partially with at least one method selected from the group consisting of enzymatic degradation, chemical degradation, physical degradation, and dissociating the template RNA and washing it away from a cDNA which has been bound to a solid phase by means of said functional group; hybridizing a reporter probe which contains all functional sequences used in an amplification reaction to at least one of said at least one further hybridization sequence of a cDNA; hybridized reporter probe is bound to a solid phase by means of said functional group of the cDNA; unbound reporter probe is removed; and at least part of said hybridized reporter probe which is the nucleic acid derived from said cDNA is amplified.

11. A method according to claim 10, wherein said reporter probe is a DNA which contains a hybridization sequence by which it is hybridized to said cDNA and a pair of hybridization sequences which will be used in the amplification reaction of step (d); and that part of said reporter probe which contains said pair of hybridization sequences is amplified in step (d) by a method selected from the group consisting of a polymerase chain reaction (PCR), a ligase chain reaction (LCR), a combination of PCR and LCR, and a multienzymatic transcription-based procedure.

12. A method according to claim 10, wherein said reporter probe is a RNA which contains a hybridization sequence by which it is hybridized to a cDNA and a pair of hybridization sequences which will be used in the amplification reaction of step (d) and a part of this reporter probe which contains said pair of hybridization sequences is amplified in step (d) by a multienzymatic transcription-based procedure.

13. A method according to claim 10, wherein said reporter probe is a primary replicative RNA which comprises a hybridization sequence by which it is hybridized to a cDNA, a RNA-ori (-) sequence extending to its 5'-end, a RNA-ori (+) sequence extending to its 3'-end and a replicase-binding domain in between; and said reporter probe is amplified in step (d) by means of a RNA replication system which comprises rNTPs in a reaction mixture and a RNA replicase that binds to the replicase binding domain of said reporter probe and replicates said reporter probe.

14. A method according to claim 10, wherein said reporter probe is at least one nucleic acid selected from the group comprising

(i) a primary replicative DNA which comprises a DNA-ori(-) sequence extending to its 5'-end, a hybridization sequence by which it is hybridized to a cDNA, and a DNA-ori(+) sequence extending to its 3'-end,

(ii) a partially double-stranded primary replicative DNA which comprises a DNA-ori(-) sequence extending to its 5'-end, a hybridization sequence by which it is hybridized to a cDNA, and a DNA-ori(+) sequence extending to its 3'-end, said 3'-terminal sequence of said reporter probe being primed with a single-stranded DNA oligonucleotide which comprises at least the first 6 nucleotides of a DNA-ori(-) sequence, and

(iii) a partially double-stranded primary replicative DNA which consists of one strand comprising a DNA-ori(-) sequence extending to the 5'-end, a hybridization sequence located further downstream which is complementary to said at least one further hybridization sequence of said cDNA, and a second hybridization sequence extending to the 3'-end to which is hybridized a second strand of DNA which contains a corresponding hybridization sequence extending to the 3'-end and a DNA-ori(-) sequence extending to the 5'-end; and said primary replicative DNA is amplified in a reaction mixture which contains dNTPs by means of a DNA replication system which involves binding of a protein primer to the DNA-ori of said primary replicative DNA and replicating said replicative DNA by means of a DNA replicase that binds to said protein-primed replicative DNA.

15. A method according to claim 1, wherein a nucleic acid amplified in step (d) is one which has been derived from said cDNA produced in step (c) by at least one further reaction selected from the group consisting of:

(i) hybridizing to said cDNA a single-stranded DNA which comprises at least a hybridization sequence which extends to the 3'-end,

(ii) synthesizing by enzymatic catalysis an at least partially double-stranded DNA,

(iii) synthesizing a RNA by enzymatic catalysis,

(iv) eliminating the template nucleic acid at least partially, and

(v) ligating the 3'-end of the cDNA with the 5'-end of another nucleic acid which is hybridized to the template nucleic acid.

16. A method according to claim 15, wherein said template nucleic acid of step (b) is RNA and contains a pair of non-overlapping hybridization sequences which will be used in the amplification reaction of step (d), at least one of these being located upstream of that hybridization sequence to which said RT primer is hybridized; said template nucleic acid further contains a P(-) sequence of a transcription promoter which is located upstream of said pair of hybridization sequences and a further hybridization sequence whose 5' boundary is located upstream of or at the 5' boundary of said P(-) sequence; said cDNA produced in step (c) contains the hybridization sequence of said RT primer, said pair of hybridization sequences, further downstream a complete P(+) sequence and said further hybridization sequence; in step (d) a nucleic acid is derived from said cDNA by

(i) eliminating the template RNA at least partially by treatment with at least one method selected from the group comprising enzymatic degradation, chemical degradation, physical degradation, and dissociating the template RNA from a solid-phase-bound cDNA and washing it away;

(ii) hybridizing a DNA oligonucleotide primer to said further hybridization sequence of the cDNA;

(iii) synthesizing by means of catalysis by a DNA-dependent DNA polymerase and dNTPs an at least partially double-stranded DNA which contains a functional transcription promoter; and

(iv) synthesizing by means of catalysis by the corresponding DNA-dependent RNA polymerase and rNTPs from said transcription promoter a RNA which contains said pair of non-overlapping hybridization sequences; said synthesized RNA, which is the nucleic acid derived from the cDNA, is amplified by means of a multienzymatic transcription-based procedure; and the reaction conditions are such that the stability of the RNA is maintained.

17. A method according to claim 15, wherein said template nucleic acid of step (b) consists of RNA and contains a further hybridization sequence located upstream of and non-overlapping with that hybridization sequence to which said RT primer is hybridized; said RT primer is a single-stranded DNA oligonucleotide, whose 5' terminal sequence consists of a DNA-ori(-) sequence corresponding to a DNA replication system; said cDNA synthesized in step (c) contains said DNA-ori(-) sequence which extends to the cDNA's 5'-end, said hybridization sequence of the RT primer and further downstream said further hybridization sequence; in step (d) a nucleic acid is derived from said cDNA by

(i) hybridizing to said further hybridization sequence of the cDNA a single-stranded DNA primer whose 5' terminal sequence consists of said DNA-ori(-) sequence; and

(ii) synthesizing by means of catalysis by a DNA-dependent DNA polymerase and dNTPs an at least partially double-stranded DNA which has a DNA-ori at least at one end and is a primary replicative DNA;

and said primary replicative DNA, which is the nucleic acid derived from said cDNA, is amplified in a reaction mixture which contains dNTPs by means of a DNA replication system which involves binding of a protein primer to the DNA-ori of said primary replicative DNA and replicating said replicative DNA by means of a DNA replicase that binds to said protein-primed replicative DNA.

18. A method according to claim 15, wherein said template nucleic acid of step (b) consists of RNA and contains a further hybridization sequence located upstream of and separated by a spacer sequence from that hybridization sequence to which said RT primer is hybridized; said RT primer is a partially double-stranded DNA adaptor of which the strand hybridized to said template nucleic acid comprises at its 5' terminal sequence the DNA-ori(-) sequence of a replicative DNA, to which a second DNA oligonucleotide is hybridized whose 3' terminal sequence consists of a DNA-ori(+) sequence; to said further hybridization sequence of the template nucleic acid a second partially double-stranded DNA adaptor is hybridized by a hybridization sequence which extends to the 5'-end of the adaptor's longer DNA strand; said second adaptor's longer strand is phosphorylated at its 5'-end and comprises a DNA-ori(+) sequence which extends to its 3'-end and to which a DNA oligonucleotide whose 5' terminal sequence consists of a complete DNA-ori(-) sequence is hybridized; said cDNA synthesized in step (c) fills the gap between these two adapters such that said cDNA is separated from said second adaptor by just one nick; in step (d) a nucleic acid is derived from said cDNA by closing said nick by means of enzymatic ligation of the 3'-end of the cDNA with the 5'-end of said second adaptor, whence a primary replicative DNA is formed; and said primary replicative DNA, which is the nucleic acid derived from the cDNA, is amplified in a reaction mixture which contains dNTPs by means of a DNA replication system which involves binding of a protein primer to the DNA-ori of said primary replicative DNA and replicating said replicative DNA by means of a DNA replicase that binds to said protein-primed replicative DNA.

19. A method according to claim 1, wherein said template nucleic acid of step (b) is RNA and contains at least one further hybridization sequence located upstream of that to which said RT primer is hybridized; said cDNA synthesized in step (c) contains said at least one further hybridization sequence; a nucleic acid amplified in step (d) is derived from said cDNA by hybridizing a reporter probe to at least one of said at least one further hybridization sequence of said cDNA and by subjecting the reaction mixture to at least one further reaction selected from the group consisting of:

(i) synthesizing a RNA by enzymatic catalysis,

(ii) eliminating the template RNA at least partially,

(iii) synthesizing an at least partially double-stranded DNA by enzymatic catalysis, and

(iv) cleaving a double-stranded DNA by enzymatic catalysis.

20. A method according to claim 19, wherein said template nucleic acid contains one further hybridization sequence that extends to its 5'-end; said further hybridization sequence consists in its 3' terminal sequence of a P(-) sequence of a transcription promoter which is upstream directly followed by at least the first three nucleotides of a 3'-RNA-ori(+) sequence; said cDNA synthesized in step (c) contains said further hybridization sequence which extends to the 3'-end; a nucleic acid amplified in step (d) is derived from said cDNA by

(i) hybridizing a reporter probe to said further hybridization sequence of the cDNA, said reporter probe being a single-stranded DNA which contains, extending to its 5'-end, the 5'-RNA-ori(-) sequence of a replicative RNA, further downstream a replicase binding domain, still further downstream a 3'-RNA-ori(+) sequence which is directly followed by the P(-) sequence of said transcription promoter; the hybridization sequence of said reporter probe consisting of said at least three last nucleotides of said 3'-RNA-ori(+) sequence and the entire P(-) sequence of said transcription promoter; the hybridization of said reporter probe to said further hybridization sequence of said cDNA constituting a functional transcription promoter, and

(ii) synthesizing from said transcription promoter a primary replicative RNA by means of catalysis by the corresponding DNA-dependent RNA polymerase and rNTPs;

and said primary replicative RNA, which is the nucleic acid derived from the cDNA, is amplified by means of an RNA replication system which comprises rNTPs in a reaction mixture and an RNA replicase that binds to the replicase binding domain of said primary replicative RNA and replicates said primary replicative RNA.

21. A method according to claim 19, wherein a nucleic acid amplified in step (d) is derived from said cDNA by

(i) eliminating the template RNA at least partially with at least one method selected from the group comprising enzymatic degradation, chemical degradation, physical degradation, and dissociating the template RNA from a cDNA by denaturation and washing it away;

(ii) hybridizing a smart probe to said at least one further hybridization sequence of said cDNA, said smart probe being a single-stranded DNA which contains, in 5' to 3' order, the P(+) sequence of a transcription promoter, a spacer sequence, a hybridization sequence complementary to said at least one further hybridization sequence of said cDNA, a further spacer sequence not complementary to the first, and the P(-) sequence of said transcription promoter directly followed by at least the first three nucleotides of a 5'-RNA-ori(-) sequence, whereby said P(-) sequence and said 5'-RNA-ori(-) nucleotides form a further hybridization sequence which extends to the 3'-end of said smart probe and is designated for the hybridization of a DNA reporter probe;

(iii) hybridizing to said further hybridization sequence of said smart probe a single-stranded DNA reporter probe which contains, in 5' to 3' order, a 5'-RNA-ori(-) sequence, a replicase binding domain, and a 3'-RNA-ori(+) sequence directly followed by the P(+) sequence of said transcription promoter, whence a functional transcription promoter is generated; and (iv) synthesizing from said transcription promoter a primary replicative RNA by means of catalysis by the corresponding DNA-dependent RNA polymerase and rNTPs; and said primary replicative RNA, which is the nucleic acid derived from the cDNA, is amplified by means of an RNA replication system which comprises rNTPs in a reaction mixture and an RNA replicase that binds to the replicase binding domain of said primary replicative RNA and replicates said primary replicative RNA.

22. A method according to claim 19, wherein said template nucleic acid of step (b) contains one further hybridization sequence which extends to its 5'-end; said cDNA synthesized in step (c) contains the corresponding further hybridization sequence extending to its 3'-end; a nucleic acid amplified in step (d) is derived from said cDNA by

(i) hybridizing a single-stranded DNA reporter probe to said further hybridization sequence of the cDNA, said reporter probe comprising upstream of a hybridization sequence by which it is hybridized to said further hybridization sequence of the cDNA multiple repetitive copies of a sequence which contains a recognition site for a restriction endonuclease;

(ii) synthesizing a double-stranded DNA by means of catalysis by a DNA-dependent DNA polymerase and dNTPs;

(iii) cleaving said double-stranded DNA by means of said restriction endonuclease, thereby generating several molecules of said double-stranded fragment; said fragment, which is the nucleic acid derived from said cDNA, is amplified in repeated cycles of denaturation, hybridization to said reporter probe, DNA synthesis, and restriction endonuclease cleavage.

23. A method according to claim 1, wherein at least one nucleic acid of the group consisting of the RT primer, a nucleic acid other than the RT primer which is hybridized to the template nucleic acid, a cDNA, a nucleic acid that is hybridized to the cDNA, a nucleic acid that is hybridized to a reporter probe, and a nucleic acid amplified in a amplification reaction carries a functional group which has at least one property selected from the group comprising being a solid phase, mediating the binding to a solid phase, being a ligand, and possessing at least one antigenic determinant.

24. A method according to claim 1 wherein, after having synthesized said cDNA in step (c), the template RNA is eliminated at least partially by treatment with at least one method selected from the group consisting of enzymatic degradation, chemical degradation, physical degradation, and dissociating the template RNA from a solid-phase-bound cDNA and washing it away.

25. A screening kit for the detection of a reverse transcriptase in accordance with the method of claim 1 comprising at least:

(i) reagents and auxiliary devices for the pretreatment of a sample including a isotonic sample dilution buffer, 0.2.mu.m filters, a reverse transcriptase extraction buffer containing salts, protein stabilizers and a mild detergent;

(ii) a RT master mixture comprising at least a buffer system, salts, a heteropolymeric template RNA, a heteropolymeric RT primer, dATP, dCTP, dGTP, and dTTP, a divalent cation selected from the group comprising Mg.sup.2+ and Mn.sup.2+, at least one protein stabilizer, a RNase inhibitor,

(iii) a RNase for the digestion of the template RNA;

(iv) at least one further oligonucleotide matched to the reverse-transcribed template RNA and reagents for a in vitro nucleic acid amplification reaction comprising at least the enzymes, nucleotide triphosphates, salts, divalent cations, and buffers;

(v) at least one positive RT control comprising a solution which contains an enzymatically active reverse transcriptase; and at least one negative RT control consisting of a solution which does not contain any enzymatically active reverse transcriptase.

26. A screening kit for the detection of a reverse transcriptase in accordance with the kit of claim 25, wherein in (iv) said reagents for an in vitro nucleic acid amplification reaction are those used in a polymerase chain reaction and comprise at least a Taq polymerase, dATP, dCTP, dGTP, dTTP, salts, a divalent cation, and buffers.